ASB9 interacts with ubiquitous mitochondrial creatine kinase and inhibits mitochondrial function

Abstract

Background: The ankyrin repeat and suppressor of cytokine signalling (SOCS) box proteins (Asbs) are a large protein family implicated in diverse biological processes including regulation of proliferation and differentiation. The SOCS box of Asb proteins is important in a ubiquitination-mediated proteolysis pathway. Here, we aimed to evaluate expression and function of human Asb-9 (ASB9).

Results: We found that a variant of ASB9 that lacks the SOCS box (ASB9DeltaSOCS) was naturally detected in human cell lines but not in peripheral blood mononuclear cells or normal hepatocytes. We also identified ubiquitous mitochondrial creatine kinase (uMtCK) as a new target of ASB9 in human embryonic kidney 293 (HEK293) cells. The ankyrin repeat domains of ASB9 can associate with the substrate binding site of uMtCK in a SOCS box-independent manner. The overexpression of ASB9, but not ASB9DeltaSOCS, induces ubiquitination of uMtCK. ASB9 and ASB9DeltaSOCS can interact and colocalise with uMtCK in the mitochondria. However, only expression of ASB9 induced abnormal mitochondrial structure and a decrease of mitochondrial membrane potential. Furthermore, the creatine kinase activities and cell growth were significantly reduced by ASB9 but not by ASB9DeltaSOCS.

Conclusions: ASB9 interacts with the creatine kinase system and negatively regulates cell growth. The differential expression and function of ASB9 and ASB9DeltaSOCS may be a key factor in the growth of human cell lines and primary cells.

Figure 1

Expression of 18 members of the ankyrin repeat and suppressor of cytokine signalling (SOCS) box protein (Asb) family in human cell lines and human peripheral blood mononuclear cells (hPBMCs). The human keratinocyte (HaCaT), human embryonic kidney 293 (HEK293), RPMI 8226, THP-1, KG-1, and human peripheral blood mononuclear cells were cultured for 1 day before total RNA isolation. The total RNAs were extracted and reverse transcribed to generate cDNAs. A total of 1 μl of the cDNA mixture was subjected to the standard PCR reaction for 25 cycles with the primer sets as described in Table 2. The β-actin expression level was used as a control. The letter M denotes a standard DNA marker.

Figure 2

Sequences of human ankyrin repeat and suppressor of cytokine signalling (SOCS) box protein 9 (ASB9), ASB9ΔSOCS and mAsb-9. CLUSTAL alignment [39] of ASB9, ASB9ΔSOCS, and mAsb-9; the location of the N-terminal ankyrin (ANK) repeats and the SOCS box motif are shown.

Figure 3

Expression patterns of human ankyrin repeat and suppressor of cytokine signalling (SOCS) box protein 9 (ASB9) and ASB9ΔSOCS. (a) The expression level of ASB9 mRNA was analysed by real-time PCR using the cDNAs from indicated human tissues. The mRNA levels were normalised using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control, and relative expression was determined by dividing all normalised values within a data set by the normalised arbitrary units of the control. (b) Expression of ASB9 in human cell lines and human peripheral blood mononuclear cells (hPBMCs). (c) Expression of ASB9 in human hepatoma cell lines and normal human hepatocytes. The expression of ASB9 mRNA and protein was analysed by means of reverse transcription (RT)-PCR and immunoblotting, respectively. The β-actin expression level was used as a control. (d) Expression of mAsb-9 in mouse cell lines and lymphoid tissues. The GAPDH expression level was used as an mRNA control. The expression of mAsb-9 protein in the mouse cell lines and primary cells was analysed by immunoblotting with anti-ASB9 antibody. The β-actin expression level was used as a protein control.

Figure 4

Identification of human ankyrin repeat and suppressor of cytokine signalling (SOCS) box protein 9 (ASB9) binding proteins. (a) Cell lysates were prepared from human embryonic kidney 293 (HEK293) cells that were stably transfected with the indicated expression vectors; immunoprecipitates were then generated by using an antibody to Myc epitope, after which they were resolved by SDS-PAGE and stained with Coomassie brilliant blue G-250. (b) The protein bands coimmunoprecipitated with ASB9 were digested in gel with trypsin, and the samples were analysed by electrospray ionization-time of flight mass spectrometry/mass spectrometry (ESI-TOF MS/MS). MS/MS analyses of the mass peaks (arrow) obtained from the 45-kDa band reveal the peptide spectra of CKB and ubiquitous mitochondrial creatine kinase (uMtCK).

Figure 5

Creatine kinase (CK)-binding specificities of human ankyrin repeat and suppressor of cytokine signalling (SOCS) box protein 9 (ASB9). (a) Expression of CKs in human normal hepatocytes human hepatoma cell lines and normal human hepatocytes. Whole lysates were subjected to immunoblotting (IB) with anti-CKB, anti-ubiquitous mitochondrial CK (uMtCK), and anti-CKM, and anti-sarcomeric MtCK (sMtCK) antibodies. The β-actin expression level was used as a control. (b) Association of ASB9 with CKB and uMtCK. The human embryonic kidney 293 calls (HEK293) cells were stably transfected with empty, Myc-tagged ASB9 and ASB9ΔSOCS expression vectors. Whole lysates or immunoprecipitates obtained with the anti-Myc antibody were subjected to immunoblotting (IB) analysis with anti-Myc, anti-CKB, anti-uMtCK, and anti-elongin B antibodies.

Figure 6

Expression and creatine kinase (CK)-binding specificities of endogenous human ankyrin repeat and suppressor of cytokine signalling (SOCS) box protein 9 (ASB9). (a) Expression of endogenous ASB9 and ASB9ΔSOCS. Cell lysates of human embryonic kidney 293 (HEK293) cells and Huh-7 cells were used to show endogenous expression of ASB9 and ASB9ΔSOCS. Endogenous expression of CKB, mitochondrial CK (MtCK), elongin B was also confirmed by immunoblotting. (b) CK-binding activity of endogenous ASB9. The immunoprecipitates obtained with the anti-ASB9 antibody were analysed by immunoblotting (IB) with anti-ASB9, anti-CKB, anti-uMtCK, and anti-elongin B antibodies.

Figure 7

Interaction of human ankyrin repeat and suppressor of cytokine signalling (SOCS) box protein (ASB9) with ubiquitous mitochondrial creatine kinase (uMtCK). (a) Schematic diagram of the ASB9 and a series of ASB9 N-terminal ankyrin (ANK) deletion mutants. (b) ANK repeats of ASB9 are required for uMtCK interaction. Myc-tagged ASB9 and a series of ANK deletion mutants were transiently cotransfected with haemagglutinin (HA)-tagged uMtCK into human embryonic kidney 293 (HEK293) cells. Cell lysates and an immunocomplex of the anti-Myc antibody were analysed by immunoblotting with anti-Myc or anti-HA antibodies.

Figure 8

Interaction of human ankyrin repeat and suppressor of cytokine signalling (SOCS) box protein 9 (ASB9) with substrate binding site of ubiquitous mitochondrial creatine kinase (uMtCK). (a) Schematic diagram of the uMtCK and an uMtCK substrate binding site (BS) deletion mutant. (b) A substrate binding site of uMtCK is required for ASB9 interaction. Haemagglutinin (HA)-tagged uMtCK or uMtCKΔBS were transiently cotransfected with Myc-tagged ASB9 or ASB9ΔSOCS into human embryonic kidney 293 (HEK293) cells, and immunocomplexing assays were performed with an anti-Myc antibody. Cell lysates and immunoprecipitates were analysed by immunoblotting with anti-Myc or anti-HA antibodies.

Figure 9

Suppressor of cytokine signalling (SOCS) box-dependent ubiquitination of creatine kinase B (CKB) and ubiquitous mitochondrial CK (uMtCK) by human ankyrin repeat and SOCS box protein 9 (ASB9). Haemagglutinin (HA)-tagged uMtCK was transiently cotransfected with Flag-ubiquitin (Ub) into HEK293 cells that stably express null (empty vector), Myc-tagged ASB9 or ASB9ΔSOCS. The cells were pretreated with MG132 for 6 h prior to the cell harvest when indicated. Cell lysates (a) and the anti-Myc antibody immunocomplexes (b) were blotted with anti-Myc, anti-Flag, anti-HA or anti-CKB antibodies.

Figure 10

In vitro ubiquitination of creatine kinase B (CKB) and ubiquitous mitochondrial CK (uMtCK) by human ankyrin repeat and suppressor of cytokine signalling (SOCS) box protein 9 (ASB9). Whole cell lysates were prepared from human embryonic kidney 293 (HEK293) cells that stably express null (empty vector), Myc-tagged ASB9 or ASB9ΔSOCS and then immunoprecipitated with anti-CKB antibody (a) or anti-uMtCK antibody (b). The immunocomplexes were analysed by means of in vitro ubiquitination assay with or without ubiquitin. The reaction mixtures were then separated by SDS-PAGE followed by immunoblotting analysis with anti-ubiquitin, anti-CKB, or anti-Myc antibodies. An immunoglobulin heavy chain is shown as an antibody amount control.

Figure 11

Degradation of creatine kinase B (CKB) and ubiquitous mitochondrial CK (uMtCK) by human ankyrin repeat and suppressor of cytokine signalling (SOCS) box protein 9 (ASB9) after long-term culture. (a) After culture of 10 passages of human embryonic kidney 293 (HEK293) cells stably transfected with Myc-tagged ASB9 and ASB9ΔSOCS expression vectors, cell lysates were immunoblotted with anti-Myc, anti-CKB, anti-uMtCK, and anti-elongin B antibodies. β-actin was used as a protein amount control. (b) The lysates obtained from HEK293 cells stably transfected with Myc-tagged ASB9 and ASB9ΔSOCS expression vectors were immunoprecipitated with an anti-Myc antibody and subjected to immunoblotting with anti-Myc, anti-CKB, anti-uMtCK, and anti-elongin B antibodies.

Figure 12

Colocalisation of human ankyrin repeat and suppressor of cytokine signalling (SOCS) box protein 9 (ASB9) and ubiquitous mitochondrial creatine kinase (uMtCK) in mitochondria. The pDSRed2-Mito vector was transfected into human embryonic kidney 293 (HEK293) cells that stably express null (empty vector), Myc-tagged ASB9 or ASB9ΔSOCS. After 24 h incubation, the cells were fixed with 4% paraformaldehyde. (a) A confocal laser scanning microscopy was used to visualise the location of individual proteins with the aid of the mouse anti-Myc antibody (for ASB9, purple) and the goat anti-uMtCK antibody (green), respectively. DSRed2-Mito was expressed in mitochondria as a red colour. Cells were stained with Hoechst no. 33258 to visualise the nuclei (blue colour). (b) A confocal laser scanning microscope was used to visualise the structure of mitochondria with the aid of DSRed2-Mito. Three-dimensional movies of the figures are available as Additional files 1 and 2.

Figure 13

Effect of human ankyrin repeat and suppressor of cytokine signalling (SOCS) box protein 9 (ASB9) on the mitochondrial membrane potential (ΔΨm). (a) Human embryonic kidney 293 (HEK293) cells that stably express null (empty vector), Myc-tagged ASB9, or ASB9ΔSOCS were trypsinised for 5 min and incubated in 50 nM tetramethylrhodamine ethyl ester (TMRE) with (full line) or without (dotted line) 200 nM carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) for 30 min at 37°C in the dark. The TMRE fluorescence in the cells was analysed by flow cytometry. The low fluorescence indicates a loss of ΔΨm. (b) The ΔΨm of peripheral blood mononuclear cells (PBMCs), RPMI 8226 cells, and KG-1 cells were analysed as described in (a).

Figure 14

Effect of human ankyrin repeat and suppressor of cytokine signalling (SOCS) box protein 9 (ASB9) on creatine kinase (CK) activity. (a) The cellular CK activity was measured during a 4-day culture of the human embryonic kidney 293 (HEK293) cells that stably express null, Myc-tagged ASB9, or ASB9ΔSOCS. The cell lysates (total cells), cytosolic fraction and mitochondrial fraction were prepared, and the CK activity was determined. (b) The cell lysates (total cells), cytosolic fraction and mitochondrial fraction of human peripheral blood mononuclear cells (hPBMCs), normal hepatocytes and human cell lines were prepared, and the CK activity was determined. (c) The immunocomplex proteins of the anti-CKB or anti-ubiquitous mitochondrial CK (uMtCK) antibodies were analysed by CK activity assay. Specific CK activity was defined in terms of units per cell (U/cell). The immunoprecipitated proteins were detected by immunoblotting analysis with an anti-CKB antibody and an anti-Myc antibody (for ASB9) (lower panel).

Figure 15

Effect of human ankyrin repeat and suppressor of cytokine signalling (SOCS) box protein 9 (ASB9) on cell growth. The cell growth was measured during a 4-day culture of the human embryonic kidney 293 (HEK293) cells that stably express null, Myc-tagged ASB9, or ASB9ΔSOCS. Cell growth was measured by means of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (a) and a trypan blue exclusion assay (b).